金属硫蛋白α和 β结构域的结构功能比较研究

金属硫蛋白具有α和β两个独立的结构域,它们结构不同,并能独立的行使功能。为了进一步研究这两个结构域之间的区别,分别采用镉和铜重组金属硫蛋白并继以枯草杆菌蛋白酶水解的方法制备α和β结构域,以及利用pGEX-4T-1这种融合表达载体表达α和β结构域。所得产物经凝胶过滤层析分离纯化后,进行了氨基酸组成,巯基和金属含量以及分子量测定,以上性质均与天然的金属硫蛋白α和β结构域相同。然后利用紫外吸收光谱和圆二色吸收光谱来研究它们的巯基金属簇结构,从UV和CD图谱可以看出,通过蛋白水解和基因表达制备的α和β结构域都具有独立的镉硫金属簇结构,但β结构域的镉硫金属簇不如α结构域紧密,其在254nm的吸收峰不象α结构域那么明显。利用DTNB的竞争反应测定了α和β结构域对镉和锌的结合力,实验结果表明,α结构域倾向于结合Cd2+,β结构域倾向于结合Zn2+。以上研究对于进一步了解α和β结构域的生理功能和分子进化提供了有利的证据。     

柱状田头菇（茶薪菇）金属硫蛋白的分离纯化与特性研究

应用快速灌注色谱系统首次从经Cd^2 诱导的柱状田头菇(茶薪菇)Agrocybe Cylindracea(DC.:Fr:)R.Maoire菌丝体中分离得到一种镉结合蛋白。通过Sephadex G-75凝胶过滤层析,原子吸收光谱分析(AAS),巯基含量测定及紫外吸收光谱分析表明这种镉结合蛋白具有金属硫蛋白(metallothionein,MT)的理化性质：即分子量为6kDa、每分子MT含18个半胱氨酸残基并结合7个镉原子、具有镉硫金属簇的特征紫外吸收光谱,初步鉴定为茶薪菇Cd—MT。     

柱状田头菇(茶薪菇)金属硫蛋白的分离纯化与特性研究

应用快速灌注色谱系统首次从经Cd2+诱导的柱状田头菇(茶薪菇)Agrocybecylindracea(DC.:Fr:)R.Maoire菌丝体中分离得到一种镉结合蛋白。通过SephadexG-75凝胶过滤层析,原子吸收光谱分析(AAS),巯基含量测定及紫外吸收光谱分析表明这种镉结合蛋白具有金属硫蛋白(metallothionein,MT)的理化性质:即分子量为6kDa、每分子MT含18个半胱氨酸残基并结合7个镉原子、具有镉硫金属簇的特征紫外吸收光谱,初步鉴定为茶薪菇Cd-MT。     

柱状田头菇(茶薪菇)金属硫蛋白的分离纯化与特性研究*

应用快速灌注色谱系统首次从经Cd2+诱导的柱状田头菇(茶薪菇)Agrocybecylindracea(DC.:Fr:)R.Maoire菌丝体中分离得到一种镉结合蛋白。通过SephadexG-75凝胶过滤层析,原子吸收光谱分析(AAS),巯基含量测定及紫外吸收光谱分析表明这种镉结合蛋白具有金属硫蛋白(metallothionein,MT)的理化性质:即分子量为6kDa、每分子MT含18个半胱氨酸残基并结合7个镉原子、具有镉硫金属簇的特征紫外吸收光谱,初步鉴定为茶薪菇Cd-MT。     

小鼠金属硫蛋白突变体αα-cDNA在烟草中的表达及转基因烟草对Cd~(2+)抗性的提高

金属硫蛋白是一类小分子量的金属结合蛋白 ,有 β,α两个结构域 .用另一个 α结构域取代金属硫蛋白的 β结构域 ,得到突变体 αα.通过 PCR法在 αα- c DNA翻译起始密码子 ATG前加入植物偏爱的碱基组合 AACA,构建 Ca MV双 35S启动子驱动的 αα- c DNA植物表达载体 p GPTVd35S-αα,此载体以抗除草剂基因 ( bar)作为选择标记物 .用根癌农杆菌介导的叶盘法对栽培种烟草 NC89进行转化 .Southern和 Western印迹分析表明突变体αα- c DNA已整合进入烟草基因组并且可以正常表达 .Northern印迹结果显示突变体αα- c DNA在烟草根部的表达强于叶部 .烟草根、茎、叶Cd2 +含量分析表明 ,转基因植物与对照相比 ,Cd2 +更多地集中在根部 ,在一定程度上降低了叶片中的 Cd2 + 含量 .Cd2 + 抗性实验进一步证明突变体αα- c DNA的表达提高了转基因烟草对 Cd2 + 的抗性 :转基因烟草在 2 0 0μmol/L Cd Cl2 条件下生长正常 ,在 40 0μmol/Cd Cl2 培养基内也可以存活 .而 1 0 0μmol/L Cd Cl2 即成为对照烟草的致死剂量 .     

鼠肝中金属硫蛋白的纯化

旨在探索鼠肝中金属硫蛋白(MT)提取工艺并加以改进。按照经典方法工艺提取MT,用中空纤维柱超滤方法加以改进,依据MT自身特点进行分离和鉴定。结果显示,粗品符合MT特点:分子量在6.5kD左右;无280nm特征吸收峰,加酸后紫外吸收(200-300nm)肩峰消失;通过离子交换可以实现MT1、MT2的分离;通过原子吸收测定,含有锌、铜、镉3种金属,锌含量最高,具有重要的病理生理意义。因此,与其它方法比较,改进后方法更适宜实验室制备。     

神经生长抑制因子的功能结构域双体

神经生长抑制因子 (neuronalgrowthinhibitoryfactor,GIF)又名金属硫蛋白 III (metallothionein III,MT III) ,是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白质 ,β结构域为其功能结构域。为深入系统地研究GIF及其结构域的结构与功能 ,构建了神经生长抑制因子功能结构域双体 (GIFβ β)。PCR扩增得到N端 β结构域和C端 β结构域的cDNA ,酶切后克隆入原核表达载体pGEX 4T 1,经发酵、诱导表达、亲和层析、凝血酶切和进一步纯化 ,每升菌液约可得重组GIFβ β蛋白 6 0mg。测其电泳行为、氨基酸组成、质谱、金属巯基含量等 ,证明得到了目的蛋白质。圆二色性图谱显示 ,GIFβ β拥有金属硫蛋白家族成员的特征———金属巯基簇结构域。MTT还原法测定神经元抑制活性大小为 :GIF >GIFβ β>GIFβ结构域     

重组金属硫蛋白HcMT的原核表达及其与金属离子竞争反应

[目的]在不同p H范围内和不同时间下检测重组HcMT结合的锌、铜离子的含量,对金属离子竞争结合金属硫蛋白的特征进行分析。[方法]构建重组表达载体,将重组质粒pET-32a-Hc MT进行原核表达,诱导时加入Zn~(2+),获得菌体后超声破碎,上清中添加Cu~(2+),分离纯化融合蛋白Trx A-Hc MT,检测金属硫蛋白溶液紫外吸收情况。样品硝化后用ICP-OES检测金属硫蛋白结合的金属离子含量。[结果]在pH 3～9的范围内,重组Hc MT上结合的Zn~(2+)含量随着p H值升高而升高。加入Cu~(2+)后,重组Hc MT上结合的Cu~(2+)浓度上升,Zn~(2+)浓度下降。在p H 3时,重组Hc MT结合金属离子的比值Cu~(2+)/Zn~(2+)为最小值0. 68,在pH 8时Cu~(2+)/Zn~(2+)为最大值1. 48,有利于Cu~(2+)竞争。当pH 8时,不同时间下Zn-Hc MT与Cu~(2+)的竞争结果表明1 h时锌铜有竞争高峰。[结论]证明了铜离子可以取代结合在重组HcMT上的锌离子,在p H 7～9范围中结合1 h竞争效率最高,该研究为重组Hc MT在重金属解毒方面的应用奠定基础。     

转金属硫蛋白突变体αα的烟草具有较高的重金属抗性

金属硫蛋白（ＭＴ）有α、β两个结构域,其中α结构域优先结合Ｃｄ＾２＋、Ｈｇ＾２＋以α结构域代替β结构域而得到的αα突变体在大肠杆菌构建及表达后,体外实验证明其具有同天然ＭＴ相似的稳定性,但结合重金属的能力稍强于天然ＭＴ。将αα突变体转入植物表达载体,以ＣａＭＶ３５Ｓ为启动子,利用根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的叶     

镉致黑斑蛙肝脏氧化损伤与金属硫蛋白含量的变化

为观察镉对黑斑蛙(Rana nigromaculata)肝脏脂质过氧化产物和金属硫蛋白含量的影响,将黑斑蛙暴露于10.0mg·mL-1浓度的镉溶液中30d,分别测定了黑斑蛙在暴露4、10d和30d时肝脏组织中镉(Cd)、还原型谷胱甘肽(GSH)、金属硫蛋白(MT)和过氧化产物丙二醛(MDA)的含量。实验结果表明,黑斑蛙肝脏中镉的积累量、GSH和MT含量均随着镉暴露时间的延长而显著升高,具有明显的时间-效应关系;在镉暴露的第10天,肝MDA含量明显高于对照组。提示镉可对黑斑蛙肝脏造成氧化损伤,而GSH、MT含量的升高则可能是机体抗氧化损伤的机理之一。     

人神经生长抑制因子β结构域的高效表达及性质研究

神经生长抑制因子（ Ｇ Ｉ Ｆ）是一种特异存在于哺乳动物脑中的金属硫蛋白（ｍ ｅｔａｌｌｏｔｈｉｏｎｅｉｎ, Ｍ Ｔ）类似物,又称 Ｍ Ｔ Ⅲ．它与 Ｍ Ｔ 有相同的结合 Ｚｎ（Ⅱ）, Ｃｄ（Ⅱ）, Ｃｕ（Ⅰ）等金属的能力,但与 Ｍ Ｔ 不同的是它能够抑制神经细胞的生长,并发现在患 Ａｌｚｈｅｉｍ ｅｒ ｄｉｓｅａｓｅ（ Ａ Ｄ 症）病人的大脑中 Ｇ Ｉ Ｆ蛋白量和ｍ Ｒ Ｎ Ａ 的量均显著下降,研究证明 Ｇ Ｉ Ｆ对神经细胞的抑制活性主要存在于其 β结构域中．为进一步研究 β结构域结构和功能的关系,将 β结构域的 ｃ Ｄ Ｎ Ａ 克隆入融合表达载体ｐ Ｇ Ｅ Ｘ ４ Ｔ １ 中, Ｉ Ｐ Ｔ Ｇ 诱导并高效表达了 β结构域蛋白,通过氨基酸组成和质谱的测定,证明得到了目的蛋白．利用金属重组的方法,分别得到了结合 Ｃｄ 和 Ｚｎ 的 Ｇ Ｉ Ｆ 的 β结构域,并测定了其巯基和金属含量对蛋白量的比值,证明所得 Ｇ Ｉ Ｆ β与 Ｍ Ｔ β在结合金属能力上十分相似．用紫外光谱学的研究表明, Ｃｄ Ｍ Ｔ 的 β结构域在２５０ ｎｍ 处比 Ｃｄ Ｇ Ｉ Ｆ 的 β结构域有一明显肩峰,从而表明二者的金属—巯基结合簇的结构有明显不同,而这种结构上的差异有可能导致二者在功能上的不同．     

脱金属硫蛋白与镉离子的络合作用及构象研究

用圆二色（ＣＤ）谱地研究兔肝脱金属硫属蛋白的两个亚型与Ｃｄ＾２＋的络合作用及对重组ＭＴ构象的影响。观测了ａｐｏ－ＭＴ垢巯基在空气和室温下的稳定性。在ＰＨ４．７１,镉重组ＭＴ１的ＣＤ谱特征峰在２５７ｎｍ（＋）,２３８ｎｍ（－）,２２６ｎｍ（＋）与镉诱导的天然ＭＴ１相同。在空气存在和ＰＨ７．９０的ＣＤ谱只有２４３ｎｍ（＋）一个峰。向两亚型分别加入７ｅｑＣｄ＾２＋测定ＣＤ谱随ＰＨ值的变化,发现在ＰＨ２．     

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., K?gi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.     

Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+

The cadmium-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-alpha MT and Zn4-alpha MT, and calf liver apo-beta MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-alpha MT, and apo-beta MT), and is observed to a much lesser extent in the titration of Zn-alpha MT. Comparison between the band centers of the Cd-alpha MT and Cd-beta MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the alpha domain, with little direct contribution from the beta domain. Analysis of the spectra recorded during titrations of Zn7-MT 2 with Cd2+ suggests: (i) that Cd2+ replaces Zn2+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspecific, "distributed" manner across both domains; (iii) that cluster formation in the alpha domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from "synthetic" Cd4,Zn3-MT following a treatment cycle that allows the redistribution of cadmium into the alpha domain; warming the synthetic "native," Cd4,Zn3-MT, to 65 degrees C results in cadmium being preferentially bound in the alpha domain; and (v) Zn7-MT will bind Cd2+ quite normally at up to 65 degrees C but with greater specificity for the alpha domain compared with titrations carried out at 25 degrees C. These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn-MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.     

岩豆凝集素分子修饰与圆二色性的关系研究

天然态岩豆凝集素（ＭＤＬ）远紫外圆二色性（ＣＤ）谱显示２１６ｎｍ处单一负峰,是一种高β－折叠构象凝集素;近紫外ＣＤ谱呈现２８２ｎｍ处负峰和２６０～２７５ｎｍ及２９５ｎｍ处的负肩,经Ｎ－乙基顺丁烯二酰亚胺（ＮＥＭＩ）和对氯汞苯甲酸（ＰＣＭＢ）修饰ＭＤＬ的巯基,其近紫外ＣＤ谱未发生变化,远紫外ＣＤ谱仅发生细微变化,ＭＤＬ凝集活性保持不变;ＰＣＭＢ过量时,ＣＤ谱呈现典型的无规卷曲谱形,ＭＤＬ完全丧失凝集活性,去除ＰＣＭＢ后,活性又全部恢复．二硫苏糖醇（ＤＤＴ）修饰ＭＤＬ的二硫键并用碘乙酸（ＩＣＨ２ＣＯＯＨ）保护巯基,ＭＤＬ远紫外ＣＤ谱２１６ｎｍ处的负峰红移至２２５ｎｍ,且显著减小;同时,近紫外ＣＤ谱２８２ｎｍ处负峰几乎消失,两负肩分别保持完整,分子中α－螺旋降低,无规卷曲增加较多,ＭＤＬ凝集活性未发生变化．用Ｎ－溴代丁二酰亚胺（ＮＢＳ）修饰ＭＤＬ分子中的色氨酸,导致２１６ｎｍ负峰蓝移至２０８ｎｍ且变小,分子中无规卷曲和α－螺旋增加,β折叠减少,近紫外ＣＤ谱２９５ｎｍ负肩消失,２８２ｎｍ负峰红移至２８７ｎｍ,ＭＤＬ凝集活性完全丧失．     

Circular dichroism of rhodopsins and porphyropsins

Circular dichroism and absorption spectra were determined for digitonin extracts of three rhodopsins: cattle, grass frog, and pigeon; and three porphyropsins: channel catfish, bluegill sunfish, and redear sunfish. A comparison of these spectra shows the following: (1) Porphyropsins, like rhodopsins, exhibit two positive CD peaks in the spectral region 321–700 nm: an α peak at about 520 nm and a small β peak at about 355 nm. These peaks substantially diminish upon bleaching. (2) In the CD spectra the α peaks of the porphyropsins are larger than the α peaks of the rhodopsins, while the β peaks are smaller than those of the rhodopsins. This is just the opposite of the corresponding relationship between the peaks in the absorption spectra. (3) The maxima of these peaks in the CD spectra of rhodopsins and porphyropsins are consistently blue-shifted from the corresponding maxima in absorption spectra. (4) Some of the visual pigments show additional positive CD peaks in the spectral region 250–320 nm. In all the visual pigments studied, the CD spectra in this region decrease on bleaching. No reciprocal relationship is observed between any of the CD bands in the visible and near ultraviolet region of the spectrum.     

Characterization of cryptomonad phycoerythrin and phycocyanin

Phycoerythrin, a chromoprotein, from the cryptomonad alga Rhodomonas lens is composed of two pairs of nonidentical polypeptides (α₂β₂). This structure is indicated by a molecular weight of 54,300, calculated from osmotic pressure measurements and by sodium dodecyl sulfate (SDS) gel electrophoresis, which showed bands with molecular weights of 9800 and 17,700 in a 1:1 molar ratio. The s_20,w⁰ of 4.3S is consistent with a protein of this molecular weight. Similar results were obtained with another cryptomonad phycoerythrin and a cryptomonad phycocyanin. Electrophoresis after partial cross-linking by dimethyl suberimidate revealed seven bands for the cryptomonad phycocyanin and six bands for cryptomonad phycoerythrin and confirmed the proposed structure. Spectroscopic studies on α and β subunits of cryptomonad phycocyanin and phycoerythrin were carried out on the separated bands in SDS gels. The individual polypeptides possessed a single absorption band with the following maxima: phycoerythrin (R. lens), α at 565 nm, β at 531 nm; phycocyanin (Chroomonas sp.), α at 644 nm, β at 566 nm. Fluorescence polarization was not constant across the visible absorption band regions of phycoerythrin (R. lens and C. ovata) with higher polarizations located at higher wavelengths, as had also been previously shown for cryptomonad phycocyanin (Chroomonas sp.). Combining the absorption spectra and the polarization results indicates that in each case the β subunit contains sensitizing chromophores and the α subunit fluorescing chromophores. The CD spectra of cryptomonad phycocyanin and both phycoerythrins were similar and were related to the spectra of the individual subunits. In Ouchterlony double-diffusion experiments the cryptomonad phycoerythrins and phycocyanins cross-reacted, with spurring, with phycoerythrin isolated from a red alga. The cryptomonad phycoerythrins were immunochemically very similar to each other and to cryptomonad phycocyanin, with little spurring detected.